Method for transferring nucleic acid into multicelled eukaryotic organism cells and combination therefor

ABSTRACT

The invention concerns an improved method for transferring in vivo multicelled eukaryotic organism cells nucleic acids or nucleic acids combined with products for enhancing the efficacy of such transfers. The invention also concerns the combination of a nucleic acid and the transfer method for use in gene therapy.

[0001] The present invention relates to a very remarkable improvement inthe in vivo transfer of nucleic acids into the cells of pluricellulareukaryotic organisms or of nucleic acids combined with products whichmake it possible to increase the yield of such transfers using weakelectric fields of between 1 and 600 V/cm, and to the combination of anucleic acid and the method of transfer according to the invention fortheir use in gene therapy.

[0002] The transfer of genes into a given cell is at the root of genetherapy. However, one of the problems is to succeed in causing asufficient quantity of nucleic acid to penetrate into cells of the hostto be treated; indeed, this nucleic acid, in general a gene of interest,has to be expressed in transfected cells. One of the approaches selectedin this regard has been the integration of the nucleic acid into viralvectors, in particular into retroviruses, adenoviruses oradeno-associated viruses. These systems take advantage of the cellpenetration mechanisms developed by viruses, as well as their protectionagainst degradation. However, this approach has disadvantages, and inparticular a risk of production of infectious viral particles capable ofdissemination in the host organism, and, in the case of retroviralvectors, a risk of insertional mutagenesis. Furthermore, the capacityfor insertion of a therapeutic or vaccinal gene into a viral genomeremains limited.

[0003] In any case, the development of viral vectors capable of beingused in gene therapy requires the use of complex techniques fordefective viruses and for complementation cell lines.

[0004] Another approach (Wolf et al. Science 247, 1465-68, 1990; Daviset al. Proc. Natl. Acad. Sci. USA 93, 7213-18, 1996) has thereforeconsisted in administering into the muscle or into the blood stream anucleic acid of a plasmid nature, combined or otherwise with compoundsintended to promote its transfection, such as proteins, liposomes,charged lipids or cationic polymers such as polyethylenimine, which aregood transfection agents in vitro (Behr et al. Proc. Natl. Acad. Sci.USA 86, 6982-6, 1989; Felgner et al. Proc. Natl. Acad. Sci. USA 84,7413-7, 1987; Boussif et al. Proc. Natl. Acad. Sci. USA 92, 7297-301,1995).

[0005] As regards the muscle, since the initial publication by J. A.Wolff et al. showing the capacity of muscle tissue to incorporate DNAinjected in free plasmid form (Wolff et al. Science 247, 1465-1468,1990), numerous authors have tried to improve this procedure (Manthorpeet al., 1993, Human Gene Ther. 4, 419-431; Wolff et al., 1991,BioTechniques 11, 474-485). A few trends emerge from these tests, suchas in particular:

[0006] the use of mechanical solutions to force the entry of DNA intocells by adsorbing the DNA onto beads which are then propelled onto thetissues (“gene gun”) (Sanders Williams et al., 1991, Proc. Natl. Acad.Sci. USA 88, 2726-2730; Fynan et al., 1993, BioTechniques 11, 474-485).These methods have proved effective in vaccination strategies but theyaffect only the top layers of the tissues. In the case of the muscle,their use would require a surgical approach in order to allow access tothe muscle because the particles do not cross the skin tissues;

[0007] the injection of DNA, no longer in free plasmid form but combinedwith molecules capable of serving as vehicle facilitating the entry ofthe complexes into cells. Cationic lipids, which are used in numerousother transfection methods, have proved up until now disappointing,because those which have been tested have been found to inhibittransfection (Schwartz et al., 1996, Gene Ther. 3, 405-411). The sameapplies to cationic peptides and polymers (Manthorpe et al., 1993, HumanGene Ther. 4, 419-431). The only case of a favourable combinationappears to be the mixing of poly(vinyl alcohol) or polyvinylpyrrolidonewith DNA. The increase resulting from these combinations only representsa factor of less than 10 compared with DNA injected in naked form(Mumper et al., 1996, Pharmaceutical Research 13, 701-709);

[0008] the pretreatment of the tissue to be injected with solutionsintended to improve the diffusion and/or the stability of DNA (Davis etal., 1993, Hum. Gene Ther. 4, 151-159), or to promote the entry ofnucleic acids, for example the induction of cell multiplication orregeneration phenomena. The treatments have involved in particular theuse of local anaesthetics or of cardiotoxin, of vasoconstrictors, ofendotoxin or of other molecules (Manthorpe et al., 1993, Human GeneTher. 4, 419-431; Danko et al., 1994, Gene Ther. 1, 114-121; Vitadelloet al., 1994, Hum. Gene Ther. 5, 11-18). These pretreatment protocolsare difficult to manage, bupivacaine in particular requiring, in orderto be effective, being injected at doses very close to lethal doses. Thepreinjection of hyperosmotic sucrose, intended to improve diffusion,does not increase the transfection level in the muscle (Davis et al.,1993).

[0009] Other tissues have been transfected in vivo either using plasmidDNA alone or in combination with synthetic vectors (reviews by Cottenand Wagner (1994), Current Opinion in Biotechnology 4, 705; Gao andHuang (1995), Gene Therapy, 2, 710; Ledley (1995), Human Gene Therapy 6,1129). The principal tissues studied were the liver, the respiratoryepithelium, the wall of the vessels, the central nervous system andtumours. In all these tissues, the levels of expression of thetransgenes have proved to be too low to envisage a therapeuticapplication (for example in the liver, Chao et al. (1996) Human GeneTherapy 7, 901), although some encouraging results have recently beenobtained for the transfer of plasmid DNA into the vascular wall (Iireset al. (1996) Human Gene Therapy 7,959 and 989). In the brain, thetransfer efficiency is very low, likewise in tumours (Schwartz et al.1996, Gene Therapy 3, 405; Lu et al. 1994, Cancer Gene Therapy 1, 245;Son et al. Proc. Natl. Acad. Sci. USA 91, 12669).

[0010] Electroporation, or use of electric fields to permeabilize cells,is also used in vitro to promote the transfection of DNA into cells inculture. However, it has up until now been accepted that this phenomenonresponded to an effect which is dependent on a threshold and that thiselectropermeabilization could only be observed for electric fields ofrelatively high intensity, of the order of 800 to 1200 volts/cm foranimal cells. This technique has also been proposed in vivo to improvethe efficacy of antitumour agents, such as bleomycin, in solid tumoursin man (American Patent No. 5, 468,228, L. M. Mir). With pulses of veryshort duration (100 microseconds), these electrical conditions (800 to1200 volts/cm) are very well suited to the intracellular transfer ofsmall molecules. These conditions (pulses of 100 microseconds) have beenapplied with no improvement for the transfer of nucleic acids in vivointo the liver, where fields of less than 1000 volts/cm have provedcompletely ineffective, and even inhibitory compared with the injectionof DNA in the absence of electrical impulses (Patent WO 97/07826 andHeller et al. FEBS Letters, 389, 225-8, 1996).

[0011] There are in fact difficulties with applying this technique invivo because the administration of fields of such an intensity may causeextensive tissue lesions to a greater or lesser extent which do notrepresent a problem for the treatment of cancer patients but which mayhave a major disadvantage for the healthy subject or the sick subjectwhen the nucleic acid is administered into tissues other than tumourtissues.

[0012] Whereas all the studies cited mention the need for high electricfields, of the order of 1000 volts/cm, to be effective in vivo, in atruly unexpected and remarkable manner, the applicants have now shownthat the transfer of nucleic acids into tissues in vivo could be verysubstantially increased, without undesirable effects, by subjecting thetissue to electrical pulses of low intensity, for example 100 or 200volts/cm and of a relatively long duration. Furthermore, the applicantshave observed that the high variability in the expression of thetransgene observed in the prior art for the transfer of DNA was notablyreduced by the method according to the invention.

[0013] Accordingly, the present invention relates to a method oftransferring nucleic acids in vivo, in which the cells of the tissuesare brought into contact with the nucleic acid to be transferred, bydirect administration into the tissue or by topical or systemicadministration, and in which the transfer is brought about byapplication to the said tissues of one or more electrical pulses of anintensity between 1 and 600 volts/cm.

[0014] According to a preferred mode, the method according to theinvention applies to tissues whose cells have specific geometries, suchas for example cells of large size and/or of elongated shape and/ornaturally responding to electrical action potentials and/or having aspecific morphology.

[0015] Preferably, the intensity of the field is between 200 and 600volts/cm and the total duration of application is greater than 10milliseconds. The number of pulses used is, for example, from 1 to100,000 pulses and the frequency of the pulses is between 0.1 and 1000Hertz. Preferably, the frequency of the pulses is between 0.2 and 100Hertz. The pulses may also be delivered in an irregular manner and thefunction which describes the intensity of the field as a function oftime may be variable. By way of example, the electric field deliveredmay result from the combination of at least one field of anintensity >400 V/cm and preferably of between 500 and 800 Volts/cm, ofshort unit duration (<1 msec), followed by one or more pulses of lowerintensity, for example <400 Volts/cm, and preferably <200 Volts/cm andof longer unit duration (>1 msec). The integral of the functiondescribing the variation of the electric field with time is greater than1 kV×msec/cm. According to a preferred mode of the invention, thisintegral is greater than or equal to 5 kV×msec/cm.

[0016] According to a preferred mode of the invention, the fieldintensity of the pulses is approximately 500 volts/cm (i.e. ±10% andpreferably ±5%).

[0017] The electrical pulses are chosen from square wave pulses,electric fields generating exponentially decreasing waves, oscillatingunipolar waves of limited duration, oscillating bipolar waves of limitedduration, or other wave forms. According to a preferred mode of theinvention, the electrical pulses are square wave pulses.

[0018] The administration of electrical pulses may be carried out by anymethod known to persons skilled in the art, for example:

[0019] system of external electrodes placed on either side of the tissueto be treated, in particular non-invasive electrodes placed in contactwith the skin,

[0020] system of electrodes implanted in the tissues,

[0021] system of electrodes/injector allowing the simultaneousadministration of the nucleic acids and the electric field.

[0022] Within the framework of the present invention, the terms transferof DNA or of nucleic acids by application of one or more electricalpulses, as well as the terms electrotransfer or alternativelyelectrotransfection should be considered as equivalent and designate thetransfer of nucleic acids or of DNA by application or in the presence ofan electric field.

[0023] The administration being carried out in vivo, it is sometimesnecessary to use intermediate products which provide electricalcontinuity with non-invasive external electrodes. This may be forexample an electrolyte in gel form.

[0024] The nucleic acids may be administered by any appropriate means,but are preferably injected in vivo directly into the tissues oradministered by another route, local or systemic and in particular bymeans of a catheter, which makes them available at the site ofapplication of the electric field. The nucleic acids may be administeredwith agents allowing or facilitating transfer, as was mentioned above.In particular, these nucleic acids may be free in solution or combinedwith synthetic agents, or carried by viral vectors. The synthetic agentsmay be lipids or polymers known to a person skilled in the art, oralternatively targeting elements allowing attachment to the membrane ofthe target tissues. Among these elements, there may be mentioned vectorscarrying sugars, peptides, antibodies or hormone receptors.

[0025] It can be understood, under these conditions of the invention,that the administration of the nucleic acids can be preceded by,simultaneous with or even subsequent to the application of the electricfields.

[0026] Accordingly, the subject of the present invention is also anucleic acid and an electric field of an intensity between 1 and 600volts/cm, as combination product for their administrationsimultaneously, separately or spaced out over time, to mammalian cellsand in particular human cells, in vivo. Preferably, the intensity of thefield is between 200 and 600 volts/cm and, more preferably still, theintensity of the field is approximately 500 volts/cm.

[0027] The method according to the present invention can be used in genetherapy, that is to say therapy in which the expression of a transferredgene, but also the modulation or the blocking of a gene, makes itpossible to provide the treatment of a particular pathologicalcondition.

[0028] Preferably, the cells of the tissues are treated for the purposeof a gene therapy allowing:

[0029] either the correction of dysfunctions of the cells themselves(for example for the treatment of diseases linked to geneticdeficiencies such as for example cystic fibrosis),

[0030] or the safeguard and/or the regeneration of the vascularizationor the innervation of the tissues or organs by trophic, neurotrophic andangiogenic factors produced by the transgene,

[0031] or the transformation of the tissue into an organ secretingproducts leading to a therapeutic effect such as the product of the geneitself (for example factors for regulation of thrombosis and ofhaemostasis, trophic factors, hormones) or such as an active metabolitesynthesized in the tissue by virtue of the addition of the therapeuticgene,

[0032] or a vaccine or immunostimulant application.

[0033] Another subject of the invention is the combination of theelectrical pulses of a field with compositions containing nucleic acidsformulated for any administration allowing access to the tissue by thetopical, cutaneous, oral, vaginal, parenteral, intranasal, intravenous,intra-arterial, intramuscular, subcutaneous, intraocular or transdermalroute, and the like. Preferably, the pharmaceutical compositions of theinvention contain a pharmaceutically acceptable vehicle for aninjectable formulation, in particular for a direct injection into thedesired organ, or for any other administration. They may be inparticular isotonic sterile solutions or dry, in particularfreeze-dried, compositions which, upon addition, depending on the case,of sterilized water or of physiological saline, allow the preparation ofinjectable solutions. The nucleic acid doses used for the injection aswell as the number of administrations and the volume of injections maybe adjusted according to various parameters, and in particular accordingto the mode of administration used, the relevant pathological condition,the gene to be expressed, or the desired duration of treatment.

[0034] The nucleic acids may be of synthetic or biosynthetic origin, ormay be extracted from viruses or prokaryotic cells or from eukaryoticcells derived from unicellular organisms (for example yeasts) or frompluricellular organisms. They may be administered in combination withall or part of the components of the organism of origin and/or of thesynthesis system.

[0035] The nucleic acid may be a deoxyribonucleic acid or a ribonucleicacid. It may be sequences of natural or artificial origin, and inparticular genomic DNA, cDNA, mRNA, tRNA and rRNA, hybrid sequences orsynthetic or semisynthetic sequences of modified or unmodifiedoligonucleotides. These nucleic acids may be obtained by any techniqueknown to persons skilled in the art, and in particular by targetinglibraries, by chemical synthesis or by mixed methods including chemicalor enzymatic modification of sequences obtained by targeting libraries.They may be chemically modified.

[0036] In particular, the nucleic acid may be a DNA or a sense orantisense RNA or an RNA having catalytic property such as a ribozyme.“Antisense” is understood to mean a nucleic acid having a sequencecomplementary to a target sequence, for example an mRNA sequence theblocking of whose expression is sought by hybridization with the targetsequence. “Sense” is understood to mean a nucleic acid having a sequencewhich is homologous or identical to a target sequence, for example asequence which binds to a protein transcription factor and which isinvolved in the expression of a given gene. According to a preferredembodiment, the nucleic acid comprises a gene of interest and elementsallowing the expression of the said gene of interest. Advantageously,the nucleic acid fragment is in the form of a plasmid.

[0037] The deoxyribonucleic acids may be single- or double-stranded, aswell as short oligonucleotides or longer sequences. They may carrytherapeutic genes, sequences for regulation of transcription or ofreplication, or regions for binding to other cellular components, andthe like. For the purposes of the invention, “therapeutic gene” isunderstood to mean in particular any gene encoding an RNA or a proteinproduct having a therapeutic effect. The protein product encoded may bea protein, a peptide and the like. This protein product may behomologous in relation to the target cell (that is to say a productwhich is normally expressed in the target cell when the latter exhibitsno pathological condition). In this case, the expression of thetransgene makes it possible, for example, to overcome an inadequateexpression in the cell or the expression of an inactive or weakly activeprotein due to a modification, or makes it possible to overexpress thesaid protein. The therapeutic gene may also encode a mutant of acellular protein having increased stability or a modified activity, andthe like. The protein product may also be heterologous in relation tothe target cell. In this case, an expressed protein may, for example,supplement or provide an activity which is deficient in the cell(treatment of enzymatic deficiencies), or may make it possible to combata pathological condition, or to stimulate an immune response for examplefor the treatment of tumours. It may be a suicide gene (Herpes ThymidineKinase) for the treatment of cancers or of restenosis.

[0038] Among the therapeutic products for the purposes of the presentinvention, there may be mentioned more particularly the genes encoding

[0039] enzymes, such as α-1-antitrypsin, proteinase (metalloproteinases,urokinase, uPA, tPA, . . . streptokinase), proteases cleaving precursorsin order to liberate active products (ACE, ICE, . . . ), or theirantagonists (TIMP-1, tissue plasminogen activator inhibitor PAI, TFPI

[0040] blood derivatives such as the factors involved in coagulation:factors VII, VIII, IX, complement factors, thrombin,

[0041] hormones, or enzymes involved in the pathway for the synthesis ofhormones, or factors involved in controlling the synthesis or theexcretion or the secretion of hormones, such as insulin, factors closeto insulin (IGF), or growth hormone, ACTH, enzymes for the synthesis ofsex hormones,

[0042] lymphokines and cytokines: interleukins, chemokines (CXC and CC),interferons, TNF, TGF, chemotactic factors or activators such as MIF,MAF, PAF, MCP-1, eotaxin, LIF, and the like (French Patent No. 9203120),

[0043] growth factors, for example IGF, EGF, FGF, KGF, NGF, PDGF, PIGF,HGF, proliferin

[0044] angiogenic factors such as VEGF of FGF, angiopoietin 1 or 2,endothelin

[0045] enzymes for synthesizing neurotransmitters,

[0046] trophic factors, in particular neurotrophic factors for thetreatment of neurodegenerative diseases, traumas which have damaged thenervous system, or retinal degeneration, such as members of the familyof neurotrophins such as NGF, BDNF, NT3, NT4/5, NT6, their derivativesand related genes—members of the CNTF families such as CNTF, axokine,LIF and derivatives thereof—IL6 and its derivatives—cardiotrophin andits derivatives—GDNF and its derivatives—members of the family of IGFs,such as IGF-1, IFGF-2 and derivatives thereof—members of the FGF family,such as FGF 1, 2, 3, 4, 5, 6, 7, 8, 9 and derivatives thereof, TGFβ

[0047] bone growth factors,

[0048] haematopoietic factors, such as erythropoietin, GM-CSF, M-CSF,LIF, and the like,

[0049] cellular architectural proteins such as dystrophin orminidystrophin (French Patent No. 91 11947),, suicide genes (thymidinekinase, cytosine deaminase, cytochrome P450-containing enzymes), genesfor haemoglobin or other protein carriers,

[0050] genes corresponding to the proteins involved in the metabolism oflipids, of the apolipoprotein type chosen from apolipoproteins A-I,A-II, A-IV, B, C-I, C-II, C-III, D, E, F, G, H, J and apo(a), metabolicenzymes such as, for example, lipases, lipoprotein lipase, hepaticlipase, lecithin-cholesterol acyltransferase, 7-alpha-cholesterolhydroxylase, phosphatidyl acid phosphatase, or alternatively lipidtransfer proteins such as the cholesterol ester transfer protein and thephospholipid transfer protein, an HDL-binding protein or alternatively areceptor chosen, for example, from the LDL receptors, the remnantchylomicron receptors and the scavenger receptors, and the like. It isfurthermore possible to add leptin for the treatment of obesity.

[0051] blood pressure regulating factors, such as the enzymes involvedin the metabolism of NO, angiotensin, bradykinin, vasopressin, ACE,renin, the enzymes encoding the mechanisms for the synthesis or for therelief of prostaglandins, thromboxan, or adenosine, adenosine receptors,kallikreins and kallistatins, ANP, ANF, diuretic or antidiureticfactors, factors involved in the synthesis, the metabolism or therelease of mediators such as histamine, serotonin, catecholamines,neuropeptides,

[0052] anti-angiogenic factors such as the ligand for Tie-1 and forTie-2, angiostatin, ATF factor, derivatives of plasminogen, endothelin,thrombospondins 1 and 2, PF-4, α- or β-interferon, interleukin-12, TNFα,urokinase receptor, flt1, KDR, PAI1, PAI2, TIMP1, prolactin fragment,

[0053] factors protecting against apoptosis, such as the AKT family,

[0054] proteins capable of inducing cell death, either active bythemselves such as the caspases or of the “pro-drug” type requiringactivation by other factors, or proteins activating pro-drugs into anagent causing cell death, such as the herpesvirus thymidine kinase,deaminases, which make it possible in particular to envisage anticancertherapies,

[0055] proteins involved in intercellular contacts and adhesion: VCAM,PECAM, ELAM, ICAM, integrins, cathenins,

[0056] proteins of the extracellular matrix,

[0057] proteins involved in the migration of cells

[0058] proteins of the signal transduction type, of the type includingFAK, MEKK, p38 kinase, tyrosines, kinases, serine-threonine kinases,

[0059] proteins involved in the regulation of the cell cycle (p21, p16,cyclines, . . . ) as well as the dominant negative mutant or derivedproteins blocking the cell cycle and capable, where appropriate, ofinducing apoptosis.

[0060] transcription factors: jun, fos, AP1, p53, . . . and the proteinsof the p53 signalling cascade.

[0061] cell structure proteins, such as the intermediate filaments(vimentin, desmin, keratins), dystrophin, the proteins involved inmuscle contractility and in controlling muscle contractibility, inparticular the proteins involved in calcium metabolism and the flow ofcalcium in the cells (SERCA, . . . ).

[0062] In the cases of proteins which function through ligand andreceptor systems, it is possible to envisage using the ligand or thereceptor (e.g. FGF-R, VEGR-R, . . . ). It is also possible to mentiongenes encoding fragments or mutants of ligand or receptor proteins, inparticular of the abovementioned proteins, either having an activitygreater than the whole protein, or an antagonist activity, or even anactivity of the “dominant negative” type relating to the initial protein(for example fragments of receptors inhibiting the availability ofcirculating proteins, associated or otherwise with sequences inducingsecretion of these fragments in relation to anchorage in the cellmembrane, or other systems for modifying the intracellular traffic ofthese ligand-receptor systems so as to divert the availability of one ofthe elements) or even possessing an inherent activity distinct from thatof the total protein (e.g. ATF).

[0063] Among the other proteins or peptides which may be secreted by thetissue, it is important to underline antibodies, the variable fragmentsof single-chain antibody (ScFv) or any other antibody fragmentpossessing recognition capacities for its use in immunotherapy, forexample for the treatment of infectious diseases, of tumours, ofautoimmune diseases such as multiple sclerosis (antiidiotype antibodies)as well as the ScFv's which becomes attached to the pro-inflammatorycytokines such as, for example, IL1 and TNFα for the treatment ofrhumatoid arthritis. Other proteins of interest are, in a nonlimitingmanner, soluble receptors such as, for example, the soluble CD4 receptoror the soluble receptor for TNF for anti-HIV therapy, the TNFα receptoror the IL1 soluble receptor for the treatment of rhumatoid arthritis,the soluble receptor for acetylcholine for the treatment of myasthenia;substrate peptides or enzyme inhibitors, or peptides which are agonistsor antagonists of receptors or of adhesion proteins such as, forexample, for the treatment of asthma, thrombosis, restenosis, metastasisor inflammation; artificial, chimeric or truncated proteins. Among thehormones of essential interest, there may be mentioned insulin in thecase of diabetes, growth hormone and calcitonin. It is also possible tomention proteins capable of inducing antitumour immunity or ofstimulating the immune response (IL2, GM-CSF, IL12, and the like).Finally, it is possible to mention the cytokines which reduce the T_(H1)response such as IL10, IL4 and Il13.

[0064] The numerous examples which precede and those which followillustrate the potential scope of the field of application of thepresent invention.

[0065] The therapeutic nucleic acid may also be an antisense sequence orgene whose expression in the target cell makes it possible to controlthe expression of genes or the transcription of cellular mRNAs. Suchsequences may, for example, be transcribed in the target cell into RNAcomplementary to cellular mRNAs and thus block their translation intoprotein, according to the technique described in European Patent No. 140308. The therapeutic genes also comprise the sequences encodingribozymes, which are capable of selectively destroying target RNAs(European Patent No. 321 201).

[0066] As indicated above, the nucleic acid may also comprise one ormore genes encoding an antigenic peptide capable of generating an immuneresponse in humans or in animals. In this particular embodiment, theinvention therefore allows either the production of vaccines, or thecarrying out of immunotherapeutic treatments applied to humans or toanimals, in particular against microorganisms, viruses or cancers. Itmay be in particular antigenic peptides specific for the Epstein-Barrvirus, the HIV virus, the hepatitis B virus (European Patent No. 185573), the pseudo-rabies virus, the “syncytia forming virus”, otherviruses or antigens specific for tumours such as the MAGE proteins(European Patent No. 259 212), such as MAGE 1, MAGE 2 proteins orantigens which can stimulate an anti-tumour response such as bacterialheat shock proteins.

[0067] Preferably, the nucleic acid also comprises sequences allowingand/or promoting the expression, in the tissue, of the therapeutic geneand/or of the gene encoding the antigenic peptide. They may be sequenceswhich are naturally responsible for the expression of the geneconsidered when these sequences are capable of functioning in thetransfected cell. They may also be sequences of different origin(responsible for the expression of other proteins, or even synthetic).In particular, they may be promoter sequences of eukaryotic or viralgenes. For example, they may be promoter sequences derived from thegenome of the cell which it is desired to transfect. Among theeukaryotic promoters, there may be mentioned any promoter or derivedsequence stimulating or repressing the transcription of a gene in aspecific manner or otherwise, strongly or weakly. They may be inparticular ubiquitous promoters (HPRT, vimentin, α-actin, tubulin, andthe like), promoters of therapeutic genes (of the type including MDR,CFTR, and the like), tissue-specific promoters (of the type includingpromoters of genes for desmin, myosins, creatine kinase,phosphoglycerate kinase) or alternatively promoters responding to astimulus such as promoters responding to the natural hormones (receptorfor steroid hormones, receptor for retinoic acid, and the like) or apromoter regulated by antibiotics (tetracyclin, rapamycin, and thelike), promoters responding to a dietary regimen such as the promotersresponding to fibrates, or other promoters responding to other moleculesof natural or synthetic origin. Likewise, they may be promoter sequencesderived from the genome of a virus. In this regard, there may bementioned, for example, the promoters of the EIA genes of theadenovirus, MLP genes, or promoters derived from genomes of the virusesCMV, RSV, SV40, and the like. The promoters may also be inducible orrepressible. In addition, these expression sequences may be modified bythe addition of activating or regulatory sequences, allowing aconditional or transient expression, a tissue-specific or predominantexpression, and the like.

[0068] Moreover, the nucleic acid may also comprise, in particularupstream of the therapeutic gene, a signal sequence directing thetherapeutic product synthesized in the secretory pathways of the targetcell. This signal sequence may be the natural signal sequence of thetherapeutic product, but it may also be any other functional signalsequence, or an artificial signal sequence. The nucleic acid may alsocomprise a signal sequence directing the synthesized therapeutic producttowards a particular compartment of the cell, such as, for example,peroxisomes, lysosomes and mitochondria for the treatment, for example,of mitochondrial genetic diseases.

[0069] Other genes which are of interest have been described inparticular by McKusick, V. A. Mendelian (Inheritance in man, catalogs ofautosomal dominant, autosomal recessive, and X-linked phenotypes. Eighthedition, John Hopkins University Press (1988)), and in Stanbury, J. B.et al. (The metabolic basis of inherited disease, Fifth edition,McGraw-Hill (1983)). The genes of interest cover the proteins involvedin the metabolism of amino acids, lipids and other constituents of thecell.

[0070] There may thus be mentioned, with no limitation being implied,the genes associated with diseases of carbohydrate metabolism such asfor example fructose-1-phosphate aldolase, fructose-1,6-diphosphatase,glucose-6-phosphatase, lysosomal α-1,4-glucosidase,amylo-1,6-glucosidase, amylo-(1,4:1,6)-transglucosidase, musclephosphorylase, muscle phosphofructokinase, phosphorylase-β-kinase,galactose-1-phosphate uridyl transferase, all the enzymes of the complexpyruvate dehydrogenase, pyruvate carboxylase, 2-oxoglutarate glyoxylasecarboxylase, D-glycerate dehydrogenase.

[0071] There may also be mentioned:

[0072] the genes associated with diseases of amino acid metabolism suchas for example phenylalanine hydroxylase, dihydrobiopterin synthetase,tyrosine aminotransferase, tyrosinase, histidinase, fumarylacetoacetase,glutathione synthetase, γ-glutamylcysteine synthetase,ornithine-δ-aminotransferase, carbamoylphosphate synthetase, ornithinecarbamoyltransferase, argininosuccinate synthetase, argininosuccinatelyase, arginase, L-lysine dehydrogenase, L-lysine ketoglutaratereductase, valine transaminase, leucine isoleucine transaminase,decarboxylase for the branched-chain 2-keto acids, isovaleryl-CoAdehydrogenase, acyl-CoA dehydrogenase, 3-hydroxy-3-methylglutaryl-CoAlyase, acetoacetyl-CoA 3-ketothiolase, propionyl-CoA carboxylase,methylmalonyl-CoA mutase, ATP:cobalamine adenosyltransferase,dihydrofolate reductase, methylenetetrahydrofolate reductase,cystathionine β-synthetase, the sarcosine dehydrogenase complex,proteins belonging to the system for cleaving glycine, β-alaninetransaminase, serum carnosinase, cerebral homocarnosinase;

[0073] the genes associated with diseases of fat and fatty acidmetabolism, such as for example lipoprotein lipase, apolipoprotein C-II,apolipoprotein E, other apolipoproteins, lecithin-cholesterolacyltransferase, LDL receptor, liver sterol hydroxylase, “phytanic acid”α-hydroxylase;

[0074] the genes associated with lysosomal deficiencies, such as forexample lysosomal α-L-iduronidase, lysosomal iduronate sulphatase,lysosomal heparan N-sulphatase, lysosomal N-acetyl-α-D-glucosaminidase,lysosomal acetyl-CoA:α-glucosamine N-acetyltransferase, lysosomalN-acetyl-α-D-glucosamine 6-sulphatase, lysosomal galactosamine6-sulphate sulphatase, lysosomal β-galactosidase, lysosomalarylsulphatase B, lysosomal β-glucuronidase,N-acetylglucosaminyl-phosphotransferase, lysosomal α-D-mannosidase,lysosomal α-neuraminidase, lysosomal aspartylglycosaminidase, lysosomalα-L-fucosidase, lysosomal acid lipase, lysosomal acid ceramidase,lysosomal sphingomyelinase, lysosomal glucocerebrosidase and lysosomalgalactocerebrosidase, lysosomal galactosylceramidase, lysosomalarylsulphatase A, α-galactosidase A, lysosomal acid β-galactosidase, αchain of lysosomal hexoaminidase A.

[0075] There may also be mentioned, in a nonrestrictive manner, thegenes associated with diseases of steroid and lipid metabolism, thegenes associated with diseases of purine and pyrimidine metabolism, thegenes associated with diseases of porphyrin and haem metabolism, thegenes associated with diseases of connective tissue, and bone metabolismas well as the genes associated with blood diseases and diseases of thehaematopoietic organs, muscle diseases (myopathy), diseases of thenervous system (neurodegenerative diseases) or diseases of thecirculatory apparatus (treatment of ischaemias and of stenosis forexample) and genes involved in mitochondrial genetic diseases.

[0076] In the method according to the invention, the nucleic acid may becombined with any type of vectors or any combination of these vectorswhich make it possible to improve the transfer of genes, for example, ina nonlimiting manner, with vectors such as viruses, synthetic orbiosynthetic agents (for example lipid, polypeptide, glycosidic orpolymeric agents), or beads which are propelled or otherwise. Thenucleic acids may also be injected into a tissue which has beensubjected to a treatment intended to improve the transfer of genes, forexample a treatment of a pharmacological nature by local or systemicapplication, or an enzymatic, permeabilizing (use of surfactants),surgical, mechanical, thermal or physical treatment.

[0077] The advantage of the use of electrotransfer in gene therapy liesin the safety provided by the local treatment linked to the use of localand targeted electric fields.

[0078] By virtue of the safety linked to the use of weak fields, thepresent invention could be applied in the region of the cardiac musclefor the treatment of cardiopathies, for example using a suitabledefibrillator. It could also be applied to the treatment of restenosisby the expression of genes inhibiting the proliferation of the smoothmuscle cells such as the GAX protein.

[0079] The combination of fields which are not very intense and whichare administered over long periods, applied to the tissues in vivo,improves the transfection of nucleic acids without causing notabledamage to the tissues. These results improve the yield of DNA transferswithin the context of gene therapy using nucleic acids.

[0080] Consequently, the method according to the invention makes itpossible, for the first time, to envisage producing, by gene therapy, anagent at physiological and/or therapeutic doses, either in the tissues,or secreted in their vicinity or into the blood stream or the lymphcirculation. Furthermore, the method according to the invention allows,for the first time, fine modulation and control of the effectivequantity of transgene expressed by the possibility of modulating thevolume of tissue to be transfected, for example with multiple sites ofadministration, or the possibility of modulating the number, the shape,the surface and the arrangement of the electrodes. An additional elementof control comes from the possibility of modulating the efficiency oftransfection by varying the field intensity, the number, the durationand the frequency of the pulses, and obviously according to the state ofthe art, the quantity and the volume of nucleic acids to beadministered. It is thus possible to obtain an appropriate transfectionlevel at the desired production or secretion level. The method finallyallows increased safety compared with the chemical or viral methods fortransferring genes in vivo, for which the affecting of organs other thanthe target organ cannot be completely excluded and controlled. Indeed,the method according to the invention allows control of the localizationof the transfected tissues (strictly linked to the volume of tissuesubjected to the local electrical pulses) and therefore provides thepossibility of a return to the initial situation by complete or partialremoval of the tissue when this is made possible by the non-vitalcharacter of this tissue and by its regeneration capacities as in thecase of the liver or the muscle. This great flexibility of use makes itpossible to optimize the method according to the animal species (humanand veterinary applications), the age of the subject, his physiologicaland/or pathological condition.

[0081] The method according to the invention makes it possible, inaddition, for the first time, to transfect nucleic acids of large sizeunlike the viral methods which are limited by the size of the capsid.This possibility is essential for the transfer of genes of a very largesize such as that for dystrophin or genes with introns and/or regulatoryelements of large size, which is necessary for example for aphysiologically regulated production of hormones. This possibility isessential for the transfer of episomes or of yeast artificialchromosomes or of minichromosomes.

[0082] The following examples are intended to illustrate the inventionin a nonlimiting manner.

[0083] In these examples, reference will be made to the followingfigures:

[0084]FIG. 1: Effects of electrical pulses of high field intensity onthe transfection of plasmid DNA pXL2774 into the cranial tibial musclein mice; mean values±SEM,

[0085]FIG. 2: Effects of electrical pulses of intermediate fieldintensity on the transfection of plasmid DNA pXL2774 into the cranialtibial muscle in mice; mean values±SEM,

[0086]FIG. 3: Effects of electrical pulses of low field intensity and ofdifferent durations on the transfection of plasmid DNA pXL2774 into thecranial tibial muscle in mice; mean values±SEM,

[0087]FIG. 4: Effects of electrical pulses of low field intensity and ofdifferent durations on the transfection of plasmid DNA pXL2774 into thecranial tibial muscle in mice; mean values±SEM,

[0088]FIG. 5: Efficiency of electrotransfection of plasmid DNA pXL2774into the cranial tibial muscle of mice at low electric fieldintensities: mean values±SEM.

[0089]FIG. 6: Map of plasmids pXL3031 and pXL3010.

EXAMPLE 1

[0090] Experiment carried out under the conditions of the prior state ofthe art in which the electric fields prove to be inhibitors oftransfection

[0091] The standard electroporation conditions, such as those used inthe prior art and which were discussed above, were tested and proved tobe ineffective, or even to have an inhibitory action on the transfer ofnucleic acids (plasmid DNA) into the striated muscle.

Materials and Methods—General Operating Conditions

[0092] In this example, the following products were used:

[0093] DNA pXL2774 (Patent PCT/FR 96/01414) is a plasmid DNA comprisingthe reporter gene for luciferase. Other products are available fromcommercial suppliers: Ketamine, Xylazine, physiological saline (0.9%NaCl).

[0094] An oscilloscope and a commercial generator of (rectangular orsquare) electrical pulses (electro-pulsator PS 15, Jouan, France) wereused. The electrodes used are flat stainless steel electrodes 5.3 mmapart.

[0095] The experiment is carried out on the mouse C57 B1/6. Mice fromdifferent cages are randomly separated before the experiment(“randomization”).

[0096] The mice are anaesthetized with a ketamine and xylazine mixture.The plasmid solution (30 μl of a solution at 500 μg/ml of 0.9% NaCl) isinjected longitudinally through the skin into the cranial tibial muscleof the left and right legs with the aid of a Hamilton syringe. The twoelectrodes are coated with a conducting gel and the injected leg isplaced between the electrodes in contact with them.

[0097] The electrical pulses are applied perpendicularly to the axis ofthe muscle with the aid of a generator of square pulses one minute afterthe injection. An oscilloscope makes it possible to control theintensity in volts (the values indicated in the examples represent themaximal values), the duration in milliseconds and the frequency in hertzof the pulses delivered, which is 1 Hz. 8 consecutive pulses aredelivered.

[0098] To evaluate the transfection of the muscle, the mice are humanelykilled 7 days after the administration of the plasmid. The cranialtibial muscles of the left and right legs are then removed, weighed,placed in lysis buffer and ground. The suspension obtained iscentrifuged in order to obtain a clear supernatant. The measurement ofthe luciferase activity is carried out on 10 μl of supernatant with theaid of a commercial luminometer in which the substrate is addedautomatically to the solution. The intensity of the luminescent reactionis given in RLU (Relative Luminescence Unit) for a muscle knowing thetotal volume of suspension. Each experimental condition is tested on 10points: 5 animals injected bilaterally. Statistical comparisons arecarried out with the aid of non-parametric tests.

Results and Discussion

[0099] Two figures, of which the scale is linear or logarithmic,illustrate the results.

[0100] In this first experiment, the effects of an electric field of 800to 1200 volts/cm which allows electroporation of tumours (Mir et al.Eur. J. Cancer 27, 68, 1991) were tested.

[0101] It is observed, according to FIG. 1, that relative to the controlgroup, where the DNA is injected without an electrical pulse:

[0102] with 8 pulses of 1200 volts/cm and of a duration of 0.1 msec, themean value of the luciferase activity is much lower,

[0103] with pulses of 1200 volts/cm and of 1 msec, 3 animals are dead,the mean value of the luciferase activity is much lower,

[0104] with pulses of 800 volts/cm and of 1 msec, the mean value of theluciferase activity is also significantly reduced.

[0105] Most of the muscles which were subjected to the action of theelectric field are visibly impaired (friable and of a whitishappearance).

EXAMPLE 2

[0106] experiment for electrotransfer of nucleic acids at moderateelectric fields

[0107] This experiment is carried out with C57 B1/6 mice. Apart from theelectric field intensity of the pulses and their duration, the practicalconditions are those of Example 1.

[0108] The results are shown in FIG. 2. The result of Example 1 isreproduced, that is to say the inhibitory effect of a series of 8 pulsesat 800 volts/cm of a duration of 1 msec on the luciferase activitydetected in the muscle. With a field of 600 volts/cm, the sameinhibition and the same impairment of the muscle tissue are observed. Onthe other hand, in a remarkable and surprising manner, the decrease involtage makes it possible to no longer visibly impair the muscles and,furthermore, at 400 and 200 volts/cm, the level of transfection of themuscles is on average greater than that obtained on the muscles notsubjected to a field. It should be noted that, relative to the controlgroup (not subjected to an electric field), the dispersion of theluciferase activity values is reduced at 200 volts/cm (SEM=20.59% of themean value against 43.32% in the absence of electric field (FIG. 2A)).

EXAMPLE 3

[0109] experiment for electrotransfer of nucleic acids with pulses oflow field intensity showing a very high stimulation of the expression ofthe transgene

[0110] This experiment is carried out with C57 B1/6 mice. Apart from theelectric field intensity of the pulses and their duration, and the factthat the pulses are delivered 25 seconds after the injection of the DNA,the practical conditions are those of the preceding examples.

[0111] The results are shown in FIG. 3. The mean value of the expressionof the luciferase transgene is markedly increased with a pulse durationof 20 msec at 100 volts/cm, and from a pulse duration of 5 msec at 200volts/cm.

[0112] This experiment also clearly shows that the mean value of theluciferase activity obtained by electrotransfection of the DNA into themuscle is a function of the duration of the electrical pulses, whenvoltages of 200 and 100 volts/cm are used. It is also observed that thedispersion of the values is notably reduced for the electrotransfectedmuscle groups (FIG. 3A). In the absence of electrical pulses (control),the SEM represents 77.43% of the mean value whereas the relative SEM ofthe mean is reduced to 14% (200 volts/cm, 5 msec), 41.27% (200 volts/cm,20 msec) and between 30% and 48% for the electrotransfer at 100 volts/cmof electric field.

[0113] Under the best condition for this experiment, the expression ofthe transgene is improved by a factor of 89.7 compared with the controlinjected in the absence of electrical pulses.

EXAMPLE 4

[0114] experiment for electrotransfer of nucleic acids into the muscleat 200 volts/cm showing an increase in the expression of the transgeneby a factor greater than 200

[0115] This experiment is carried out in DBA 2 mice, with electricalpulses of a field intensity of 200 volts/cm and of variable duration,the other conditions of this experiment being those of Example 3.

[0116] This example confirms that at 200 volts/cm, the transfection ofthe luciferase activity is increased from a pulse duration of 5 msec andthen continues to increase for longer durations (FIGS. 4 and 5). Hereagain, a reduction in the inter-individual variability indicated by theSEM relative to the non-electrotransfected control (the relative valueof the SEM is equal to 35% for the control and 25, 22, 16, 18, 16 and26% for series of pulses of 1, 5, 10, 15, 20 and 24 msec respectively),is observed with electrotransfection.

[0117] Under the best condition for this experiment, the expression ofthe transgene is improved by a factor of 205 relative to the controlinjected in the absence of electrical pulses.

EXAMPLE 5

[0118] efficiency of the electrotransfer of nucleic acids as a functionof the product “number of pulses×field intensity×duration of each pulse”

[0119]FIG. 5 exemplifies the importance of the parameter correspondingto the product “number of pulses×field intensity×duration of eachpulse”. This parameter in fact corresponds to the integral, as afunction of time, of the function which describes the variation of theelectric field.

[0120] The representation in FIG. 5 of the results obtained duringexperiments 2, 3 and 4 with electric field intensities of 200 V/cm, 100V/cm or in the absence of electric fields shows that the transfectionefficiency increases as a function of the product of the total durationof exposure to the electric field by the field intensity. A stimulatingeffect is obtained for a value greater than 1 kV×msec/cm of the product“electric field×total duration of the pulses”. According to a preferredmode, a stimulation is obtained for a value greater than or equal to 5kV×msec/cm of the product “electric field×total duration of the pulses”.

[0121] In the following examples, the electrotransfer of nucleic acidsby means of the method according to the invention was tested on varioustumours, either of human origin implanted on nude (immunodeficient) miceor of murine origin implanted on C57B1/6 (immunocompetent) mice.

EXAMPLE 6

[0122] experiment for electrotransfer of nucleic acids into humanpulmonary tumours H1299

[0123] The experiment is carried out in 18 to 20 g female nude mice. Themice are monolaterally implanted with grafts of H1299 tumours of 20 mm³.The tumours develop, reaching a volume of 200 to 300 mm³. The mice aresorted according to the sizes of their tumours and divided intohomogeneous groups. The mice are anaesthetized with a Ketamine, Xylazinemixture. The plasmid solution (40 μl of a solution at 250 μg/ml of DNAin 20 mM NaCl, 5% glucose) is longitudinally injected at the centre ofthe tumour with the aid of a Hamilton syringe. The side faces of thetumour are coated with conducting gel and the tumour is placed betweenthe two electrodes. The electrodes are stainless steel plate electrodes0.45 to 0.7 cm apart. An oscilloscope and a commercial generator of(rectangular or square) electrical pulses (electro-pulsator PS 15,Jouan, France) were used.

[0124] In this example, the plasmid used is plasmid pXL3031 (FIG. 6)containing the gene encoding luciferase (cytoplasmic). The plasmidpXL3031 is a vector derived from the vector pXL2774 (WO 97/10343) intowhich the luc+gene encoding modified Photinus pyralis luciferase(cytoplasmic) obtained from pGL3basic (Genbank: CVU47295) has beenintroduced under the control of the promoter obtained from the humancytomegalovirus early region (hCMV IE, Genbank HS5IEE) and thepolyadenylation signal of the SV40 virus late region (Genbank SV4CG).

[0125] The electrical pulses are applied with the aid of a square pulsegenerator 20 to 30 sec after the injection. An oscilloscope makes itpossible to control the intensity in Volts, the duration in millisecondsand the frequency in hertz of the pulses delivered, that is to say 200to 800 Volts/cm, 20 msec and 1 hertz.

[0126] For the evaluation of tumour transfection, the mice (10 mice percondition) are humanely killed 2 days after the injection of theplasmid. The tumours are removed, weighed and ground in a lysis buffer.The suspension obtained is centrifuged in order to obtain a clearsupernatant. The luciferase activity is measured in 10 μl of supernatantwith the aid of a commercial luminometer in which the substrate is addedautomatically. The results are expressed in total RLU (Relative lightUnit) per tumour.

[0127] In this example, two series of experiments were carried out inorder to determine the effect of the electric field intensity on theefficiency of the transfection into the human pulmonary tumours H1299.In a first series of experiments, electric field intensities of 200 to500 Volts/cm were tested. In a second series of experiments, electricfield intensities varying from 400 to 800 Volts/cm were tested. TABLE 1Effect of electrical pulses of different field intensities on thetransfection of plasmid DNA pXL 3031 on human tumors H1299 (non-smallcell pulmonary carcinomas); mean values +/− SEM for the luciferaseactivity in RLU per tumour. Conditions: electric field intensity V/cm asindicated in the table, 8 pulses of 20 msec, frequency 1 Hertz.Experiment 1 Experiment 2 Volt/cm Mean SEM Mean SEM 0 32.8 ±6.8 44.7±10.2 200 129.7 ±39.1 300 585.0 ±134.8 400 5,266.6 ±1,473.8 8,488.2±3,881.7 500 14,201.6 ±6,162.6 600 7,401.0 ±5,323.1 800 11,884.1±4,048.3

[0128] It is observed, according to Table 1, that relative to thecontrol group where the DNA is injected without electrical pulse, thegene transfer is increased in a manner dependent on the electric fieldintensity of 200 to 400 Volts/cm to reach a plateau corresponding to themaximum transfection obtained from 500 volts/cm. At higher voltages (600and 800 volts/cm), skin or deeper burns are obtained without, however,reducing the expression of the transgene.

[0129] The amplification of the gene transfer obtained byelectrotransfer into the pulmonary tumours H1299 is of the order of 240to 320 fold.

EXAMPLE 7

[0130] Experiment for electrotransfer of nucleic acids into human colontumours HT29

[0131] The experiment is carried out in 18 to 20 g female nude mice. Themice are monolaterally implanted with grafts of tumours HT29 of 20 mm³.The tumours develop, reaching a volume of 100 to 200 mm³. The mice aresorted according to the size of their tumours and divided intohomogeneous groups. Apart from the distance used between the electrodes(0.45 cm), the implementation conditions are those of Example 6. Theresults of two series of independent experiments are presented in Table2. TABLE 2 Effect of electrical pulses of different field intensities onthe transfection of plasmid DNA pXL 3031 on human tumours HT29 (colonadenocarcinomas); mean values +/− SEM for the luciferase activity in RLUper tumour. Conditions: electric field intensity V/cm as indicated inthe table, 8 pulses of 20 msec, frequency 1 Hertz. Experiment 1Experiment 2 Volt/cm Mean SEM Mean SEM 0 4.0 ±1.8 0.6 ±0.3 400 16.0 ±5.4500 14.1 ±7.6 5.5 ±3.6 600 24.2 ±9.2 14.6 ±6.4

[0132] Compared with the control groups without electrotransfer, theapplication of an electric field of an intensity of 600 volts/cm makesit possible to reach an optimum level of transfection regardless of thebase level of transfection without electrotransfer. The improvement inthe transfection is by a factor of 6 to 23 fold respectively, and isrelatively similar from 400 to 600 Volts/cm.

EXAMPLE 8

[0133] Experiment for electrotransfer of nucleic acids into murinefibrosarcomas

[0134] The experiment is carried out in 18 to 20 g C57B1/6 mice. Themice are monolaterally implanted with 1×10⁶ LPB cells in 100 μl ofserum-free MEM medium. The tumours develop, reaching a volume of 100 to200 mm³. The mice are sorted according to the size of their tumours anddivided into homogeneous groups. The conditions for carrying out theexperiment are those of Example 6.

[0135] The results of two series of independent experiments arepresented in Table 3. TABLE 3 Effect of electrical pulses of differentfield intensities on the transfection of plasmid DNA pXL 3031 on murinefibrosarcomas; mean values +/− SEM for the luciferase activity in RLUper tumour. Conditions: electric field intensity V/cm as indicated inthe table, 8 pulses of 20 msec, frequency 1 Hertz. Experiment 1Experiment 2 Volt/cm Mean SEM Mean SEM 0 0.6 ±0.3 0.4 ±0.1 300 26.3±14.8 11.6 ±4.6 400 42.5 ±31.2 10.4 ±3.5 500 17.0 ±12.8 6.0 ±1.8 60011.0 ±7.1

[0136] Compared with the control groups without electrotransfer, theapplication of an electric field of an intensity of 300 to 600 Volts/cmmakes it possible to improve the gene transfer by a factor of 30 to 70,regardless of the applied voltage.

EXAMPLE 9

[0137] Experiment for electrotransfer of nucleic acids into murine B16melanomas

[0138] The experiment is carried out in 18 to 20 g C57B1/6 mice. Themice are monolaterally implanted with grafts of B16 tumours of 20 mm³.The tumours develop, reaching a volume of 200 to 300 mm³. The mice aresorted according to the size of their tumours and divided intohomogeneous groups.

[0139] The conditions for carrying out the experiment are those ofExample 6.

[0140] The results are presented in Table 4. TABLE 4 Effect ofelectrical pulses of different field intensities on the transfection ofplasmid DNA pXL 3031 murine B16 melanomas; mean values +/− SEM for theluciferase activity in RLU per tumour. Conditions: electric fieldintensity V/cm as indicated in the table, 8 pulses of 20 msec, frequency1 Hertz. Experiment 1 Volt/cm Mean SEM 0 1.3 ±0.7 300 14.3 ±7.6 500 32.2±12.6 600 17.2 ±6.2

[0141] Compared with the control group without electrotransfer, theapplication of an electrical field of an intensity of 500 Volts/cm makesit possible to improve the gene transfer by a factor of 24.

EXAMPLE 10

[0142] Experiment for electrotransfer of nucleic acids into murine 3LLtumours

[0143] The experiment is carried out in 18 to 20 g C57B1/6 mice. Themice are monolaterally implanted with grafts of 3LL tumours of 20 mm³.

[0144] The size of the transfected tumours obtained five days after theimplantation is 30 mm³. The conditions for carrying out the experimentare those of Example 6. The results are presented in Table 5. TABLE 5Effect of electrical pulses of different field intensities on thetransfection of plasmid DNA pXL 3031 on murine 3LL pulmonary carcinomas;mean values +/− SEM for the luciferase activity in RLU per tumour.Conditions: electric field intensity V/cm as indicated in the table, 8pulses of 20 msec, frequency 1 Hertz. Volt/cm Mean SEM 0 0.1 ±0.04 3003.7 ±2.9 500 470.5 ±237.6 600 53.3 ±23.9

[0145] The application of an electric field of an intensity of 500Volts/cm makes it possible to increase the expression of the transgeneby a factor of 3885.

[0146] These remarkable results should be related to the fact that thesetumours are only very slightly transfectable with DNA when the DNA issimply injected without electrotransfer.

EXAMPLE 11

[0147] Experiment for electrotransfer of nucleic acids into humanpulmonary tumours H1299, effect on the secretion into the plasma of thesecreted human alkaline phosphatase.

[0148] In this example, the DNA pXL3010 (FIG. 6) used is a plasmid DNAcontaining the gene encoding the secreted placental human alkalinephosphatase.

[0149] The plasmid pXL3010 is a vector derived from ColE1 into which thegene encoding the secreted alkaline phosphatase obtained frompSEAP-basic (Clontech, Genbank: CVU09660) has been introduced under thecontrol of the CMV promoter obtained from the plasmid pCDNA3(Invitrogen, the Netherlands) and of the polyadenylation signal of theSV40 virus late region (Genbank SV4CG).

[0150] The experiment is carried out in 18 to 20 g nude mice. The miceare monolaterally implanted with grafts of H1299 tumours of 20 mm³. Thetumours develop, reaching a volume of 200 to 300 mm³. The mice aresorted according to the size of their tumours and divided intohomogeneous groups.

[0151] The tumours are transfected under the implementation conditionsof Example 6 with, however, a single voltage condition, that is to say500 Volts/cm, 20 msec and 1 hertz.

[0152] The assays of alkaline phosphatase are carried out in the plasmawith the aid of the Phospha-light kit (Tropix) on day D1, D2 and D8after the transfection with or without electrotransfer. The results arepresented in Table 6. TABLE 6 Effect of electrical pulses of differentfield intensities on the secretion of an exogeneous protein: humanalkaline phosphatase secreted following transfection of plasmid DNA pXL3010 in human tumours H1299; mean values +/− SEM for the alkalinephosphatase (ng/ml). Conditions: electrical field intensity V/cm asindicated in the table, 8 pulses of 20 msec, frequency 1 Hertz. Alkalinephosphatase in the plasma Sample 0 Volt/cm 500 Volts/cm collection (MOY+/− SEM) (MOY +/− SEM) D1 1.42 ± 0.07 8.90 ± 1.74 D2 1.40 ± 0.01 9.04 ±1.55 D8 1.31 ± 0.01 1.67 ± 0.12

[0153] All the results presented in Examples 6 to 11 show that theelectrotransfer of nucleic acids under the conditions of the methodaccording to the invention makes it possible to remarkably increase thelevel of expression of the transgene, in various types of tumours.Furthermore, in the case of transgene encoding a secreted protein, theintratumour administration of the plasmid by electrotransfer makes itpossible to significantly increase the plasma concentration of thesecreted protein.

EXAMPLE 12

[0154] Effect of the increase of the duration of the electrical pulses

[0155] This example illustrates that it is possible to increase the unitduration of the pulses well above the values tested in Example 4.

[0156] This experiment is carried out with C57B1/6 mice. The plasmidused is the plasmid pXL2774, the quantity of DNA administered is 15 μg.The electropulsator used to deliver the electrical pulses of a durationgreater than 20 msec is a commercial electropulsator (Genetronics, modelT 820, USA, San Diego, Calif.). The electrical pulses are variable innumber and duration but have a constant field intensity of 200 Volts/cm;the other conditions for this experiment are those described inExample 1. The results are presented in Table 7. TABLE 7 Mean values +/−SEM for the luciferase activity in millions of RLU per muscle. N = 10for each group. Electrotransfer conditions: field intensity 200 V/cm, 8or 4 pulses (variable unit duration), frequency 1 Hz. Pulse duration(msec) 0 1 5 10 20 30 40 50 60 80 Experiment A 11 ± 5 39 ± 6 211 ± 26288 ± 46 1158 ± 238 1487 ± 421 2386 ± 278 8 pulses Experiment A 11 ± 526.8 ± 6   123 ± 17 246 ± 32  575 ± 88  704 ± 130 3440 ± 1077 4 pulsesExperiment B 15 ± 8 2885 ± 644 2626 ± 441 1258 ± 309 4 pulse

[0157] Table 7: Mean values±SEM for the luciferase activity in millionsof RLU per muscle. N=10 for each group. Electrotransfer conditions:field intensity 200 V/cm, 8 or 4 pulses (variable unit duration),frequency 1 Hz.

[0158] An increase in the expression of the transgene is observed withthe extension of the unit duration of the pulses (at least up to 40 msecfor a series of 8 pulses and at least up to 50 msec for a series of 4pulses of an intensity of 200 Volts/cm). This example shows that theoptimum duration of the pulses depends on the number of pulses used andthat the unit duration of the pulses may reach at least 80 msec, thisvalue for the duration not being limiting.

EXAMPLE 13

[0159] Efficiency of electrotransfer as a function of the number ofelectrical pulses

[0160] This example demonstrates the effect of the increase in thenumber of electrical pulses on the efficiency of the transfer of nucleicacids.

[0161] This experiment is carried out with C57B1/6 mice. The plasmidused is the plasmid pXL 2774, the quantity of DNA administered is 15 μg.The electrical pulses are variable in number. The duration of each pulseis 20 msec. The field intensity is 200 Volts/cm. The other conditionsfor this experiment are those described in Example 1. The results arepresented in Table 8. TABLE 8 Mean value +/− SEM for the luciferaseactivity in millions RLU per muscle. N = 10 per group. Conditions: fieldintensity 200 V/cm, variable number of pulses of 20 msec, frequency 1Hz. Number of 0 1 2 4 6 8 12 16 pulses Total RLU 70 ± 147 ± 281 ± 439 ±678 ± 819 ± 929 ± 890 ± 56 26 46 50 129 73 169 137

[0162] It is observed that the expression of the luciferase increasessubstantially from the application of a single pulse, and that itcontinues to increase as a function of the number of pulses. It thusappears that the variation of the number of pulses delivered is a meansof modulating the efficiency of the transfer of nucleic acids and ofadjusting the level of expression of the transgene.

[0163] The reduction in the variability of the response demonstrated bythe reduction in the value of the SEM relative to the mean for all thegroups subjected to the electrotransfer is also confirmed.

EXAMPLE 14

[0164] Effect of the increase in the frequency of the electrical pulses.

[0165] This example shows that the increase in the frequency of thepulses unexpectedly makes it possible to improve the efficiency of thetransfection. Moreover, and in a clinical perspective, the increase inthe frequency must improve the comfort of the patient by reducing thetotal duration of the treatment.

[0166] This experiment is carried out with C57B1/6 mice. The plasmidused is the plasmid pXL 2774, the quantity of DNA administered is 15 μg.The frequency of the electrical pulses is variable (from 0.1 to 4Hertz). The duration of each pulse is 20 msec, the field intensity is200 Volts/cm, the other conditions for this experiment are thosedescribed in Example 1. The results are presented in Table 9. TABLE 9Mean values +/− SEM for the luciferase activity in millions of RLU permuscle. N = 10 for each group. Conditions: field intensity 200 V/cm, 8or 4 pulses of 20 msec, variable frequency. Frequency 0 0.1 0.2 0.5 1 23 4 Hertz Experiment A 5 ± 54 ± 95 ± 405 ± 996 ± 1528 ± 8 pulses 2 13 1660 156 257 Experiment A 114 ± 163 ± 175 ± 337 ± 587 ± 4 pulses 14 24 2653 90 Experiment B 21 ± 1294 ± 2141 ± 3634 ± 2819 ± 8 pulses 14 189 387868 493 Experiment B 1451 ± 1572 ± 1222 ± 2474 ± 4 pulses 228 320 126646

[0167] The results obtained in experiment “A”, Table 9 show that thehighest frequencies (≧1 Hz) are more effective than the lowerfrequencies which correspond to a longer duration between twoconsecutive pulses (10 seconds at 0.1 Hertz). The transfectionefficiency increases with the frequency over the range of values testedfrom 0.1 to 4 Hertz for 4 pulses and from 0.1 to 3 Hertz for 8 pulses.

EXAMPLE 15

[0168] Effect of the application of an electric field varying accordingto a decreasing exponential as a function of time.

[0169] This example demonstrates the effect of the application of anelectric field varying according to a decreasing exponential on theefficiency of the transfer of nucleic acids.

[0170] This experiment is carried out with C57B1/6 mice.

[0171] The plasmid used is the plasmid pXL 3031. The plasmid pXL3031(FIG. 12) is a vector derived from the plasmid pXL2774 (WO 97/10343)into which the luc+gene encoding the modified Photinus pyralisluciferase (cytoplasmic) obtained from pGL3basic (Genbank: CVU47295) hasbeen introduced under the ocntrol of a promoter obtained from the humancytomegalovirus early region (hCMV IE, Genbank HS5IEE) and of thepolyadenylation signal of the SV40 virus late region (Genbank SV4CG).The quantity of DNA administered is 10 μg.

[0172] The electrical pulse generator used makes it possible to deliverpulses of an electric field intensity varying according to a decreasingexponential as a function of time (Equibio electropulsator, modeleasyjectT plus, Kent UK). The voltage imposed is the exponential peakvoltage. The second adjustable parameter is the capacitance (μFarads)which makes it possible to vary the quantity of energy delivered and theexponential time constant. The results are presented in Table 10. TABLE10 Factor of increase in expression (luciferase activity) obtained byapplication of an exponentially decreasing pulse. The increase factor iscalculated with reference to the luciferase activity obtained with theadministration of the plasmid pXL3031 without electrotransfer. (Meanvalues of the increase factor, N = 4 to 6 per condition). Capa Capa CapaCapa Capa Capa Capa μF μF μF μF μF μF μF 150 300 450 600 1200 2400 3000 40 V/cm 1.23 11 100 V/cm 16.5 2.8 6.5 23.9 150 V/cm 1.8 3.5 6.1 200V/cm 5.1 15.8 18.8 121.5 189.7 300 V/cm 32.1 90.5 48.7 760.4 56.2 400V/cm 795 600 V/cm 62 800 V/cm 3.1 1.1

[0173] By way of comparison, the factor of increase in expressionobtained for the transfer of pXL3031 in the presence of an electricfield with square-shaped pulses (field intensity of 200 V/cm, 8 pulsesof 20 msec, at a frequency of 1 Hertz) was 44 in the same experiment.

[0174] These results show that it is possible to use electrical pulsesof square shape or of exponentially decreasing intensity as a functionof time. Furthermore, in the latter case, a substantial increase inexpression may be obtained for a low field value and a high capacitance(e.g. 200 V/cm, capacitance 3000 μFarad) or a high field value and a lowcapacitance (e.g. 400 V/cm, capacitance 300 μFarad).

EXAMPLE 16

[0175] Effect of the combination of a brief pulse of high voltage and ofseveral long pulses of low voltage.

[0176] This example shows that the electric field delivered may be acombination of at least one field between 500 and 800 Volta/cm for ashort duration, for example 50 or 100 μsec, and of at least one weakfield (<100 Volts/cm) for a longer duration, for example ≧1 msec and upto 90 msec in this experiment.

[0177] The low electric field values here are 80 V/cm applied at 4pulses of a duration of 90 msec with a frequency of 1 Hertz. For thisexperiment, two electropulsators are used. The electrical pulses areapplied by one and then the other apparatus, the change being made inless than one second with the aid of a manual control.

[0178] The plasmid used is the plasmid pXL3031. The quantity of DNAadministered is 3 μg. The electric field values are indicated in Table11; the other conditions for this experiment are those described inExample 1. TABLE 11 Mean values +/− SEM for the luciferase activity inmillions of RLU per muscle. N = 10 muscles per group. Conditions forapplication of the Experiment 1 Experiment 2 electric field (3 μgpXL3031) (3 μg pXL3031) Control (absence of  320 +/− 126  75 +/− 27electric field) A1: 500 V/cm, — 169 +/− 63 1 × 0.1 msec A3: 800 V/cm, 416 +/− 143 272 +/− 84 1 × 0.1 msec B: 80 V/cm, 1282 +/− 203 362.21 +/−85.17 4 × 90 msec Conditions A1 then B — 1479 +/− 276 Conditions A3 thenB 3991 +/− 418 1426 +/− 209 Conditions B then A3 — 347 +/− 66

[0179] Table 11, summarizing the results obtained for two series ofexperiments, shows that a brief pulse of high voltage or that foursuccessive long pulses of low voltage only slightly improve thetransfection compared with the control group which received an injectionof pXL3031 but was not subjected to an electric field. The same applieswhen the low field pulses are applied before the high field pulse.

[0180] On the other hand, in the two series of experiments, thecombination of a brief pulse of high voltage followed by four successivelong pulses of low voltage very considerably increases the efficiency ofthe transfer of the DNA.

[0181] The results obtained in Examples 1 and 2 showed that 8 pulses of600, 800 or 1200 volts of a unit duration of 1 msec at 1 Hz causedlesions and inhibited transfection. The results obtained in Example 16show that, under particular conditions, it is possible to use highvoltage field intensities in a manner which does not cause lesions;indeed, from a macroscopic point of view, the muscles are never visiblyimpaired. The use of high electric fields of brief duration combinedwith low fields of longer duration appears as an additional means ofmodulating the efficiency of the transfer of the DNA.

EXAMPLE 17

[0182] Effect of the time of injection of the nucleic acid relative tothe application of the electric field.

[0183] This example illustrates the fact that the nucleic acid may beadministered at least 30 minutes, and even at least one hour, before theapplication of the electric field.

[0184] This experiment is carried out with C57B1/6 mice. The plasmidused is the plasmid pXL 2774. The quantity of DNA administered is 15 μgor 1.5 μg. The injection of DNA is followed, or preceded, by theapplication of an electric field under the following conditions:intensity 200 V/cm, 8 pulses of 20 msec, frequency 1 Hz. The otherconditions for this experiment are those described in Example 1. Acontrol group consists of animals which received an injection of theplasmid but were not subjected to the electrical pulses. The results arepresented in Table 12. TABLE 12 Mean values +/− SEM for the luciferaseactivity in millions of RLU per muscle. N = 10 muscles per group. Table12A: Injection of DNA in the absence of electric field Exp 1 Exp 2 Exp 3Exp 4 Exp 5 pXL2774 pXL 2774 pXL 2774 pXL 2774 pXL 2774 (15 μg) (15 μg)(1.5 μg) (15 μg) (1.5 μg) Control 7 ± 4 8 ± 6 0.4 ± 0.2  22 ± 15  1 ± 1Table 12B: Injection of DNA before application of the electric fieldtime Exp 1 Exp 2 Exp 3 Exp 4 Exp 5 −120 min 20 ± 5  1 ± 1  −60 min 106 ±22 10 ± 3  −30 min 303 ± 36  237 ± 61  7 ± 3 184 ± 22 15 ± 4  −5 min 410± 7   −60 sec 253 ± 51   −20 sec 492 ± 122 201 ± 43  9 ± 3 123 ± 23 12 ±2 Table 12C: Injection of DNA after application of the electric fieldtime Exp 1 Exp 2 Exp 3 Exp 4 Exp 5 +10 sec  7 ± 7 +20 sec 11 ± 6  0.4 ±0.1 +60 sec 8 ± 7  17 ± 15

[0185] The presence of the DNA at the time of the application of theelectric field is a condition for the efficiency of theelectrotransfection. Remarkably, it is observed that the injection ofthe plasmid may be carried out at least 30 minutes and even 1 hour(Experiments 4 and 5) before the application of the electric field,without notably modifying the level of expression. A similar result isobtained both with with a dose of 15 μg of plasmid per muscle and with a10-fold lower dose of 1.5 μg.

[0186] These observations make it possible in particular to envisagemultiple injections, at variable times, of the same plasmid, or ofdifferent plasmids into the muscle prior to the application of theelectric field. It is also possible to make multiple injections over anextended region of the muscle and then to apply a series of electricalpulses over the entire injected territory to be treated.

EXAMPLE 18

[0187] Transfer of a gene encoding erythropoietin (EPO)

[0188] Adult C57B1/6 mice received, in the cranial tibial muscle andunilaterally, an injection of plasmid pXL3348. The plasmid pXL3348 (FIG.16) is a vector derived from the plasmid pXL2774 into which the murinegene for erythropoietin (NCBI: 193086) has been introduced under thecontrol of the promoter obtained from the human cytomegalovirus earlyregion (hCMV IE) and of the polyadenylation signal of the SV40 viruslate region (Genbank SV4CG).

[0189] The conditions for electrotransfer are the following: electricfield intensity 200 V/cm, 8 pulses of 20 msec, frequency 1 Hz. Theelectric field is applied imemdiately after injection of the plasmidDNA. TABLE 13 Mean values ± SEM. N = 4 to 5. Serum erythropoietin Serumerythropoietin (mIU/ml) (mIU/ml) at D7 at D24 Electro- Electro- Electro-Electro- transfer transfer transfer transfer Plasmid − + − + pXL3348 03.0 ± 1.6 0 1.12 ± 0.8  (1 μg) pXL3348 0.9 ± 0.9 61.8 ± 15.8 0 74.1 ±28.9 (10 μg) pUCl9 0 0 (1 μg) Haematocrit % Haematocrit % samplecollection at D7 Sample collection at D24 Electro- Electro- Electro-Electro- transfer transfer transfer transfer Plasmid − + − + pXL334838.5 ± 0.5 35.0 ± 3.6 50.8 ± 2.3  81 ± 0.5 (1 μg) pXL3348 32.0 ± 3.226.0 ± 4.1 69.0 ± 5.1 83.0 ± 1.0 (10 μg) PUC 19 30.8 ± 2.3 43.2 ± 0.9 (1μg)

[0190] With electrotransfer, a very clear increase in the quantity oferythropoietin in the blood at D7 and D24 is observed for theadministration of 10 μg of pXL3348. Furthermore, the physiologicaleffect of the increase in erythropoietin which results in an increase inthe haematocrit is very high (85%), from D7, even for a very smallquantity of plasmid (1 μg).

EXAMPLE 19

[0191] Effect of the electrotransfer on the expression of vaccinaltransgenes

[0192] This example demonstrates that the method according to theinvention is also applicable to the transfer of genes encodingpolypeptides of vaccinal importance.

[0193] The experiment is carried out in 9-week old female Balb/c mice.The electrodes used are stainless steel plate electrodes 5 mm apart.VR-HA is a plasmid DNA containing the haemagglutinin gene of theinfluenza virus (strain A/PR/8/34). VR-gB is a plasmid DNA containingthe gene for glycoprotein B (gB) of the human cytomegalovirus (Townestrain).

[0194] The plasmid solution (50 μl of a solution at 20 μg/ml or 200 μ/mlin 0.9% NaCl) is longitudinally injected through the skin into thecranial tibial muscle unilaterally. The electrical pulses are applied 20sec after the administration of the plasmid, perpendicularly to the axisof the muscle with the aid of the square pulse generator (electric fieldintensity 200 V/cm, 8 consecutive pulses of a duration of 20 msec,frequency 1 Hz).

[0195] For the evaluation of the stimulation of the immune response, thefollowing immunization protocol was followed: D 0 collection of thepreimmune serum D 1 primary injection, plus or minus electrotransfer D 2collection of the immune serum D 2 injection of booster, plus or minuselectrotransfer D 42 Collection of immune serum D 63 collection ofimmune serum

[0196] The blood samples are collected at the level of the retro-orbitalsinus. The assays of the specific antibodies are carried out by ELISA.Each experimental condition is tested on 10 animals injectedunilaterally.

[0197] The results relating to the titres of antibodies directed againstthe influenza virus haemagglutinin are presented in Table 14 A. Electro-transfer D0 D21 D42 D63 VR-HA − <50 132 ± 739 1201 ± 4380 1314 ± 2481 (1μg) VR-HA + <50 1121 ± 1237 10441 ± 7819  8121 ± 5619 (1 μg) (p)(0.0135) (0.0022) (0.0033) VR-HA − <50 781 ± 666  5113 ± 16015 4673 ±8238 (10 μG) VR-HA + <50 4153 ± 2344 74761 ± 89228 41765 ± (10 μG) 52361(p) (0.0002) (0.0005) (0.0007)

[0198] Table 14-a: Titres of antibodies directed against the influenzavirus haemagglutinin, obtained after injection of 1 or 10 μg of DNA(VR-HA) in the absence or in the presence of electrical pulses. Theresults are the geometric means for 10 animals (8 animals for the groupinjected with 1 μg of DNA in the presence of electrical pulses andsamples collected at D63)±standard deviation. The value of p wasobtained by comparing in pairs the groups injected in the presence andin the absence of electrical pulses using the Man-Whitney nonparametrictest.

[0199] These results show that the titres of antibodies directed againstthe influenza virus haemagglutin are increased by a factor of about 10in the groups subjected to electrical pulses. Thus, the mice whichreceived 1 μg of DNA in the presence of electrical pulses have a meanantibody titre slightly greater than that of the mice which received 10μg of DNA in the absence of electrical pulse.

[0200] The results relating to the titres of antibodies directed againstthe human cytomegalovirus glycoprotein B are presented in Table 14 B.TABLE 14 Titres of antibodies directed Electro- transfer D 0 D 21 D 42 D63 VR-gB − <50  73 ± 138  755 ± 1766 809 ± 1363 (10 μg) VR-gB + <50 200± 119 3057 ± 1747 2112 ± 1330 (10 μg) (p) (0.0558) (0.0108) (0.0479)

[0201] Table 14B: Titres of antibodies directed against the humancytomegalovirus glycoprotein B (gB), obtained after injection of 10 μgof DNA (VR-gB) in the absence or in the presence of electrical pulses.The results are the geometrical means for 10 animals (9 animals for thegroup injected in the presence of electrical pulses)±standard deviation.The value of p was obtained by comparing in pairs the groups injected inthe presence and in the absence of electrical pulses using theMan-Whitney nonparametric test.

[0202] These results show that the titres of antibodies directed againstthe human cytomegalovirus glycoprotein B are increased by a factor of 4at D42, in the group subjected to the electrical pulses. It is alsonoted that the coefficient of variation is on average three times lowerin the groups of animals subjected to the electrical pulses.

1. Method of transferring nucleic acid into cells of pluricellulareukaryotic organisms in vivo in which the cells of the tissue arebrought into contact with the nucleic acid to be transferred by directadministration into the tissue or by topical or systemic administrationand in which the transfer is brought about by application to the saidtissues of one or more electrical pulses of an intensity of between 1and 600 volts/cm.
 2. Method according to claim 1, characterized in thatthe cells of the tissues have specific geometries (large size and/orelongated shape and/or naturally corresponding to electrical actionpotentials and/or having a specific morphology).
 3. Method according toclaim 1, characterized in that the intensity of the field is between 200and 600 volts/cm.
 4. Method according to claim 1, characterized in thatthe intensity of the field is approximately 500 volts/cm.
 5. Methodaccording to one of claims 1 to 4, characterized in that the totalduration of application of the electric field is greater than 10milliseconds.
 6. Method according to one of claims 1 to 5, characterizedin that the application, to the tissue, of the electric field comprisesone or more pulses of regular frequency.
 7. Method according to claim 6,characterized in that the application, to the tissue, of the electricfield comprises between 1 and 100,000 pulses of frequency between 0.1and 1000 hertz.
 8. Method according to one of claims 1 to 5,characterized in that the electrical pulses are delivered in anirregular manner relative to each other and in that the functiondescribing the intensity of the electric field as a function of the timefor one pulse is variable.
 9. Method according to one of claims 1 to 8,characterized in that the integral of the function describing thevariation of the electric field with time is greater than 1 kV×msec/cm.10. Method according to claim 9, characterized in that this integral isgreater than or equal to 5 kV×msec/cm.
 11. Method according to one ofclaims 1 to 10, characterized in that the electrical pulses are chosenfrom square wave pulses, electric fields generating exponentiallydecreasing waves, oscillating unipolar waves of limited duration,oscillating bipolar waves of limited duration, or other wave forms. 12.Method according to one of claims 1 to 11, characterized in that theelectrical pulses comprise square wave pulses.
 13. Method according toone of claims 1 to 12, characterized in that the electrical pulses areapplied using electrodes placed on either side of the tissue to betreated or placed in contact with the skin.
 14. Method according to oneof claims 1 to 12, characterized in that the electrical pulses areapplied using electrodes introduced inside the tissue to be treated. 15.Method according to one of claims 1 to 14, characterized in that thenucleic acid is injected into the tissue.
 16. Method according to one ofclaims 1 to 14, characterized in that the nucleic acid is injected bythe systemic route.
 17. Method according to claim 16, characterized inthat the nucleic acid is injected by the intra-arterial or intravenousroute.
 18. Method according to one of claims 1 to 14, characterized inthat the nucleic acid is administered by the topical, cutaneous, oral,vaginal, intranasal, subcutaneous or intraocular route.
 19. Methodaccording to one of claims 1 to 18, characterized in that the nucleicacid is present in a composition containing, in addition,pharmaceutically acceptable excipients for the different modes ofadministration.
 20. Method according to claim 19, characterized in thatthe composition is suitable for parenteral administration.
 21. Methodaccording to one of claims 1 to 20, characterized in that the nucleicacid is a deoxyribonucleic acid.
 22. Method according to one of claims 1to 20, characterized in that the nucleic acid is a ribonucleic acid. 23.Method according to one of claims 1 to 22, characterized in that thenucleic acid is of synthetic or biosynthetic origin, or extracted from avirus or from a unicellular or pluricellular eukaryotic or prokaryoticorganism.
 24. Method according to claim 23, characterized in that thenucleic acid administered is combined with all or part of the componentsof the organism of origin and/or of the synthesis system.
 25. Methodaccording to one of claims 1 to 24, characterized in that the nucleicacid encodes an RNA or a protein of interest.
 26. Method according toclaim 25, characterized in that the RNA is a catalytic or antisense RNA.27. Method according to claim 25, characterized in that the nucleic acidencodes a protein chosen from enzymes, blood derivatives, hormones,lymphokines, cytokines, growth factors, trophic factors, angiogenicfactors, neurotrophic factors, bone growth factors, haematopoieticfactors, coagulation factors, antigens and proteins involved in themetabolism of amino acids, lipids and other essential constituents ofthe cell.
 28. Method according to claim 27, characterized in that thenucleic acid encodes the angiogenic factors VEGF and FGF, theneurotrophic factors BDNF, CNTF, NGF, IGF, GMF, aFGF, NT3, NT5, the Gaxprotein, growth hormone, α-1-antitrypsin, calcitonin, leptin and theapolipoproteins, the enzymes for the biosynthesis of vitamins, hormonesand neuromediators.
 29. Method according to claim 25, characterized inthat the nucleic acid codes for an antibody, a variable fragment ofsingle-chain antibody (ScFv) or any other antibody fragment possessingrecognition capacities for the purposes of immunotherapy, or codes for asoluble receptor, a peptide which is an agonist or antagonist of areceptor or of an adhesion protein, for an artificial, chimeric ortruncated protein.
 30. Method according to claim 29, characterized inthat the nucleic acid encodes an antiidiotype antibody, a solublefragment of the CD4 receptor or of the TNFa receptor or of theacetylcholine receptor.
 31. Method according to one of claims 27 to 30,characterized in that the nucleic acid encodes a precursor of atherapeutic protein.
 32. Method according to one of claims 1 to 31,characterized in that the nucleic acid is in the form of a plasmid. 33.Method according to one of claims 1 to 31, characterized in that thenucleic acid contains a gene of large size and/or introns and/orregulatory elements of small or large size.
 34. Method according to oneof claims 1 to 31, characterized in that the nucleic acid is an episomalDNA or a yeast artificial chromosome or a minichromosome.
 35. Methodaccording to one of claims 1 to 34, characterized in that the nucleicacid contains sequences allowing and/or promoting the expression of thetransgene in the tissue.
 36. Method according to one of claims 1 to 35,characterized in that the acid is combined with any type of vectors orwith any combination of vectors which make it possible to improve thetransfer of nucleic acid, such as viruses, synthetic or biosyntheticagents, or beads which are propelled or otherwise.
 37. Method accordingto one of claims 1 to 36, characterized in that the tissue is subjectedto a treatment intended to improve gene transfer, a treatment ofpharmacological nature in the form of a local or systemic application,or an enzymatic, permeabilizing, surgical, mechanical, thermal orphysical treatment.
 38. Method according to one of claims 1 to 36,characterized in that it makes it possible to cause the tissue toproduce an agent at physiological and/or therapeutic doses, either inthe muscle cells, or secreted.
 39. Method according to one of claims 1to 38, characterized in that it makes it possible to modulate thequantity of transgene expressed by modulating the volume of tissuetransfected.
 40. Method according to claim 39, characterized in that itmakes it possible to modulate the volume of tissue transfected by theuse of multiple sites of administration.
 41. Method according to one ofclaims 1 to 40, characterized in that it makes it possible to modulatethe quantity of transgene expressed by modulating the number, shape,surface and arrangement of the electrodes, and by varying the fieldintensity, the number, the duration, the frequency and the form of thepulses, as well as the quantity and the volume of nucleic acid foradministration.
 42. Method according to one of claims 1 to 41,characterized in that it makes it possible to control the localizationof the tissues transfected by the volume of tissue subjected to thelocal electrical pulses.
 43. Method according to one of claims 1 to 42,characterized in that it allows a return to the initial situation byremoval of the transfected tissue area.
 44. Nucleic acid and electricfield of an intensity between 1 and 600 volts/cm, as combination productfor their administration separately or spaced out over time in vivo to atissue, and for gene therapy based on in vivo electrotransfection intothe tissues after administration of the nucleic acid.
 45. Combinationproduct according to claim 44, characterized in that the field intensityis between 200 and 600 volts/cm.
 46. Combination product according toclaim 44, characterized in that the field intensity is approximately 500volts/cm.
 47. Combination product according to one of claims 44 to 46,characterized in that the total duration of application of the electricfield is greater than 10 milliseconds.
 48. Combination product accordingto one of claims 44 to 47, characterized in that the application, to thetissue, of the electric field comprises one or more pulses of regularfrequency.
 49. Combination product according to claim 48, characterizedin that the application, to the tissue, of the electric field comprisesbetween 1 and 100,000 pulses of frequency between 0.1 and 1000 hertz.50. Combination product according to one of claims 44 to 47,characterized in that the electrical pulses are delivered in anirregular manner relative to each other and in that the functiondescribing the intensity of the electric field as a function of time forone pulse is variable.
 51. Combination product according to one ofclaims 44 to 50, characterized in that the integral of the functiondescribing the variation of the electric field with time is greater than1 kV×msec/cm.
 52. Combination product according to claim 51,characterized in that this integral is greater than or equal to 5kV×msec/cm.
 53. Combination product according to one of claims 44 to 52,characterized in that the electrical pulses are chosen from square wavepulses, electric fields generating exponentially decreasing waves,oscillating unipolar waves of limited duration, oscillating bipolarwaves of limited duration, or other wave forms.
 54. Combination productaccording to one of claims 44 to 53, characterized in that theelectrical pulses comprise square wave pulses.
 55. Combination productaccording to one of claims 44 to 54, characterized in that theelectrical pulses are applied externally.
 56. Combination productaccording to one of claims 44 to 54, characterized in that theelectrical pulses are applied inside the tissue.
 57. Combination productaccording to one of claims 44 to 56, characterized in that the nucleicacid is injected into the tissue.
 58. Combination product according toone of claims 44 to 56, characterized in that the nucleic acid isinjected by the systemic route.
 59. Combination product according toclaim 58, characterized in that the nucleic acid is injected by theintra-arterial or intravenous route.
 60. Combination product accordingto one of claims 44 to 56, characterized in that the nucleic acid isadministered by the topical, cutaneous, oral, vaginal, intranasal,intramuscular, subcutaneous or intraocular route.
 61. Combinationproduct according to one of claims 44 to 60, characterized in that thenucleic acid is present in a composition containing, in addition,pharmaceutically acceptable excipients for the different modes ofadministration.
 62. Method according to claim 61, characterized in thatthe composition is suitable for parenteral administration. 63.Combination product according to one of claims 44 to 62, characterizedin that the nucleic acid is a deoxyribonucleic acid.
 64. Combinationproduct according to one of claims 44 to 62, characterized in that thenucleic acid is a ribonucleic acid.
 65. Combination product according toone of claims 44 to 64, characterized in that the nucleic acid is ofsynthetic or biosynthetic origin, or extracted from a virus or aunicellular or pluricellular eukaryotic or prokaryotic organism. 66.Combination product according to claim 65, characterized in that thenucleic acid administered is combined with all or part of the componentsof the organism of origin and/or of the synthesis system. 67.Combination product according to one of claims 44 to 66, characterizedin that the nucleic acid encodes an RNA or a protein of interest. 68.Combination product according to claim 67, characterized in that the RNAis a catalytic or antisense RNA.
 69. Combination product according toclaim 67, characterized in that the nucleic acid encodes a proteinchosen from enzymes, blood derivatives, hormones, lymphokines, growthfactors, trophic factors, angiogenic factors, neurotrophic factors, bonegrowth factors, haematopoietic factors, coagulation factors, antigensand proteins involved in the metabolism of amino acids, lipids and otheressential constituents of the cell.
 70. Combination product according toclaim 69, characterized in that the nucleic acid encodes the angiogenicfactors VEGF and FGF, the neurotrophic factors BDNF, CNTF, NGF, IGF,GMF, aFGF, NT3, NT5, the Gax protein, growth hormone, a cytokine,α-1-antitrypsin, calcitonin, leptin and the apolipoproteins, the enzymesfor the biosynthesis of vitamins, hormones and neuromediators. 71.Combination product according to claim 67, characterized in that thenucleic acid codes for an antibody, a variable fragment of single-chainantibody (ScFv) or any other antibody fragment possessing recognitioncapacities for the purposes of immunotherapy, or codes for a solublereceptor, a peptide which is an agonist or antagonist of a receptor orof an adhesion protein, for an artificial, chimeric or truncatedprotein.
 72. Combination product according to claims 71, characterizedin that the nucleic acid encodes an antiidiotype antibody, a solublefragment of the CD4 receptor or of the TNFa receptor or of theacetylcholine receptor.
 73. Combination product according to one ofclaims 69 to 72, characterized in that the nucleic acid encodes aprecursor of a therapeutic protein.
 74. Combination product according toone of claims 44 to 73, characterized in that the nucleic acid is in theform of a plasmid.
 75. Combination product according to one of claims 44to 73, characterized in that the nucleic acid contains a gene of largesize and/or introns and/or regulatory elements of small or large size.76. Combination product according to one of claims 44 to 73,characterized in that the nucleic acid is an episomal DNA or a yeastartificial chromosome or a minichromosome.
 77. Combination productaccording to one of claims 44 to 76, characterized in that the nucleicacid contains sequences allowing and/or promoting the expression of thetransgene in the tissue.
 78. Combination product according to one ofclaims 44 to 77, characterized in that the acid is combined with anytype of vectors or with any combination of vectors which make itpossible to improve the transfer of nucleic acid, such as viruses,synthetic or biosynthetic agents, or beads which are propelled orotherwise.
 79. Combination product according to one of claims 44 to 78,characterized in that the tissue is subjected to a treatment intended toimprove gene transfer, a treatment of pharmacological nature in the formof a local or systemic application, or an enzymatic, permeabilizing,surgical, mechanical, thermal or physical treatment.
 80. Combinationproduct according to one of claims 44 to 79, characterized in that itmakes it possible to cause the tissue to produce an agent atphysiological and/or therapeutic doses, either in the cells of thetissue, or secreted.
 81. Combination product according to one of claims44 to 79, characterized in that it makes it possible to modulate thequantity of transgene expressed by modulating the volume of tissuetransfected.
 82. Combination product according to claim 81,characterized in that it makes it possible to modulate the volume oftissue transfected by the use of multiple sites of administration. 83.Combination product according to one of claims 44 to 82, characterizedin that it makes it possible to modulate the quantity of transgeneexpressed by modulating the number, shape, surface and arrangement ofthe electrodes, and by varying the field intensity, the number, theduration, the frequency and the form of the pulses, as well as thequantity and the volume of nucleic acid for administration. 84.Combination product according to one of claims 44 to 83, characterizedin that it makes it possible to control the localization of the tissuestransfected by the volume of tissue subjected to the local electricalpulses.
 85. Combination product according to one of claims 44 to 84,characterized in that it allows a return to the initial situation byremoval of the transfected tissue area.
 86. Use of nucleic acid for themanufacture of a medicament for the treatment, by gene therapy, of cellsor of tissues of pluricellular eukaryotic organisms by bringing the saidcells or tissues into contact with the nucleic acid to be transferred invivo and then applying to the said cells or tissues one or moreelectrical pulses of an intensity of between 1 and 800 volts/cm.
 87. Useaccording to claim 86, characterized in that the bringing into contactis carried out by direct administration into the cells or the tissues orby topical or systemic administration.
 88. Use according to either ofclaims 86 and 87, characterized in that the waves are unipolar.
 89. Useaccording to one of claims 86 to 88, in which the intensity of the fieldis between 4 and 400 volts/cm.